Abstract

Advanced breast carcinoma is a main cause of mortality affecting women. It is associated with poor prognosis; therefore, understanding the molecular mechanisms of invasive malignancies is critical in order to discover new therapies and to optimise current therapies. Degradation of the Extracellular Matrix (ECM) is a crucial step in tumour growth and invasion, allowing the cancerous cells to metastasize to distant organs and form secondary tumours. Matrix metalloproteinases (MMP) are a family of zinc-dependant endopeptidases that have the capability to degrade the ECM, enabling tumours cells to invade and migrate. Hence, MMP overexpression and/or enhanced activity are positively associated with breast cancer metastasis and invasion. Two MMPs have been shown to be involved in breast cancers, gelatinase A (MMP-2) and gelatinase B (MMP-9). The activity of MMPs is tightly regulated by endogenous inhibitors that are called tissue inhibitors of metalloproteinases (TIMPs). There are four different endogenous negative regulator proteins of MMPs, with TIMP-1 and TIMP-2 inhibiting MMP-9 and -2, respectively. Due to their potential role in tumour metastasis, a great interest has grown in developing novel methods to inhibit the MMPs as there is a direct relation between the expression of MMPs and the invasiveness of the tumours. The Farnesoid X Receptor (FXR) is a nuclear receptor that is highly expressed in breast cancers, and has been reported to be involved in in regulation of MMP and TIMP activity in hepatic and vascular tissues. The rationale of the current study was to investigate whether FXR is a novel regulator of matrix metalloprotease-2 and -9 in metastatic breast cancer cells, and hence may represent a novel therapeutic target. Two FXR agonists were used to measure their effects on breast cancer cells MDA-MB-231, MDA-MB-468 (triple negative), MCF-7 (Estrogen receptor positive) and normal cells MCF10A: chenodeoxycholic acid (CDCA) is an endogenous (low-affinity, low-selectivity) ligand, while 3-[2-[2-Chloro-4-[[3-(2, 6-dichlorophenyl)-5-(1-methylethyl)-4isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064) is a synthetic (high affinity, high-selectivity) ligand. Cell viability, protein and mRNA levels of matrix metalloprotease -2 and -9 and TIMP-2 and -1, and cell migration were assessed using both molecular and cellular techniques. Both FXR agonists decreased breast cancer and normal breast cell viability, with the effects more significant in the triple negative cells, suggesting a potential targeting towards aggressive, hard-to-treat cancer cell-types. However, the FXR ligands didn’t alter mRNA and protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 intracellularlly or extracellularlly, suggesting that this cytotoxic effect may not be via MMP. FXR ligands also had no effect on breast cancer cell migration, which is consistent with the suggestion that FXR activation has a general cytotoxic effect on tumour cells, but does not directly impact on tumour migration. In conclusion, FXR activation does not impact on markers of breast cancer metastasis, suggesting that its potential as a therapeutic target to prevent tumour progression may be limited. However, the cytotoxic effect on cancer cells is promising, suggesting that these agonists may still possess some potential for breast cancer therapy.